Substrate requirements for duplex DNA translocation by the eukaryal and archaeal minichromosome maintenance helicases

Replicative DNA helicases are ring-shaped hexamers that play an essential role in DNA synthesis by separating the two strands of chromosomal DNA to provide the single-stranded (ss) substrate for replicative polymerases. Biochemical and structural studies suggest that these helicases translocate along one strand of the duplex, which passes through and interacts with the central channel of these ring-shaped hexamers, and displace the complementary strand. A number of these helicases were shown to also encircle both strands simultaneously and then translocate along double-stranded (ds)DNA. In this report it is shown that the Schizosaccharomyces pombe Mcm4,6,7 complex and archaeal minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus move along duplex DNA. These two helicases, however, differ in the substrate required to support dsDNA translocation. Although the S. pombe Mcm4,6,7 complex required a 3'-overhang ssDNA region to initiate its association with the duplex, the archaeal protein initiated its transit along dsDNA in the absence of a 3'-overhang region, as well. Furthermore, DNA substrates containing a streptavidin-biotin steric block inhibited the movement of the eukaryotic helicase along ss and dsDNAs but not of the archaeal enzyme. The M. thermautotrophicus MCM helicase, however, was shown to displace a streptavidin-biotin complex from ss, as well as dsDNAs. The possible roles of dsDNA translocation by the MCM proteins during the initiation and elongation phases of chromosomal replication are discussed.     

DnaB drives DNA branch migration and dislodges proteins while encircling two DNA strands

DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling one DNA strand. This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding. Thus, DnaB may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo. DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases.     

Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.     

Unraveling DNA helicases. Motif, structure, mechanism and function.

DNA helicases are molecular 'motor' enzymes that use the energy of NTP hydrolysis to separate transiently energetically stable duplex DNA into single strands. They are therefore essential in nearly all DNA metabolic transactions. They act as essential molecular tools for the cellular machinery. Since the discovery of the first DNA helicase in Escherichia coli in 1976, several have been isolated from both prokaryotic and eukaryotic systems. DNA helicases generally bind to ssDNA or ssDNA/dsDNA junctions and translocate mainly unidirectionally along the bound strand and disrupt the hydrogen bonds between the duplexes. Most helicases contain conserved motifs which act as an engine to drive DNA unwinding. Crystal structures have revealed an underlying common structural fold for their function. These structures suggest the role of the helicase motifs in catalytic function and offer clues as to how these proteins can translocate and unwind DNA. The genes containing helicase motifs may have evolved from a common ancestor. In this review we cover the conserved motifs, structural information, mechanism of DNA unwinding and translocation, and functional aspects of DNA helicases.     

Mechanochemistry of t7 DNA helicase

The bacteriophage T7 helicase is a ring-shaped hexameric motor protein that unwinds double-stranded DNA during DNA replication and recombination. To accomplish this it couples energy from the nucleotide hydrolysis cycle to translocate along one of the DNA strands. Here, we combine computational biology with new biochemical measurements to infer the following properties of the T7 helicase: (1) all hexameric subunits are catalytic; (2) the mechanical movement along the DNA strand is driven by the binding transition of nucleotide into the catalytic site; (3) hydrolysis is coordinated between adjacent subunits that bind DNA; (4) the hydrolysis step changes the affinity of a subunit for DNA allowing passage of DNA from one subunit to the next. We construct a numerical optimization scheme to analyze transient and steady-state biochemical measurements to determine the rate constants for the hydrolysis cycle and determine the flux distribution through the reaction network. We find that, under physiological and experimental conditions, there is no dominant pathway; rather there is a distribution of pathways that varies with the ambient conditions. Our analysis methods provide a systematic procedure to study kinetic pathways of multi-subunit, multi-state cooperative enzymes.     

On translocation mechanism of ring-shaped helicase along single-stranded DNA

The ring-shaped helicases represent one important group of helicases that can translocate along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA by using the energy derived from NTP binding and hydrolysis. Despite intensive studies, the mechanism by which the ring-shaped helicase translocates along ssDNA and unwinds dsDNA remains undetermined. In order to understand their chemomechanical-coupling mechanism, two models on NTPase activities of the hexamers in the presence of DNA have been studied here. One model is assumed that, of the six nucleotide-binding sites, three are noncatalytic and three are catalytic. The other model is assumed that all the six nucleotide-binding sites are catalytic. In terms of the sequential NTPase activity around the ring and the previous determined crystal structure of bacteriophage T7 helicase it is shown that the obtained mechanical behaviors such as the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle using the former model are in good quantitative agreement with the previous experimental results for T7 helicase. Moreover, the acceleration of DNA unwinding rate with the stimulation of DNA synthesis by DNA polymerase can also be well explained by using the former model. In contrast, the ssDNA-translocation size and DNA-unwinding size per dTTPase cycle obtained by using the latter model are not consistent with the experimental results for T7 helicase. Thus it is preferred that the former model is the appropriate one for the T7 helicase. Furthermore, using the former model some dynamic behaviors such as the rotational speeds of DNA relative to the T7 helicase when translocation along ssDNA and when unwinding dsDNA have been predicted, which are expected to test in order to further verify the model.     

The replicative helicases of bacteria, archaea, and eukarya can unwind RNA-DNA hybrid substrates

Replicative helicases are hexameric enzymes that unwind DNA during chromosomal replication. They use energy from nucleoside triphosphate hydrolysis to translocate along one strand of the duplex DNA and displace the complementary strand. Here, the ability of a replicative helicase from each of the three domains, bacteria, archaea, and eukarya, to unwind RNA-containing substrate was determined. It is shown that all three helicases can unwind DNA-RNA hybrids while translocating along the single-stranded DNA. No unwinding could be observed when the helicases were provided with a single-stranded RNA overhang. Using DNA, RNA, and DNA-RNA chimeric oligonucleotides it was found that whereas the enzymes can bind both DNA and RNA, they could translocate only along DNA and only DNA stimulates the ATPase activity of the enzymes. Recent observations suggest that helicases may interact with enzymes participating in RNA metabolism and that RNA-DNA hybrids may be present on the chromosomes. Thus, the results presented here may suggest a new role for the replicative helicases during chromosomal replication or in other cellular processes.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

DNA Unwinding by Ring-Shaped T4 Helicase gp41 Is Hindered by Tension on the Occluded Strand

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, ‘occluded’ strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.     

Non-hexameric DNA helicases and translocases: mechanisms and regulation

Helicases and nucleic acid translocases are motor proteins that have essential roles in nearly all aspects of nucleic acid metabolism, ranging from DNA replication to chromatin remodelling. Fuelled by the binding and hydrolysis of nucleoside triphosphates, helicases move along nucleic acid filaments and separate double-stranded DNA into their complementary single strands. Recent evidence indicates that the ability to simply translocate along single-stranded DNA is, in many cases, insufficient for helicase activity. For some of these enzymes, self assembly and/or interactions with accessory proteins seem to regulate their translocase and helicase activities.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

Mcm4,6,7 uses a "pump in ring" mechanism to unwind DNA by steric exclusion and actively translocate along a duplex

Mcm4,6,7 is a ring-shaped heterohexamer and the putative eukaryotic replication fork helicase. In this study, we examine the mechanism of Mcm4,6,7. Mcm4,6,7 binds to only one strand of a duplex during unwinding, corresponding to the leading strand of a replication fork. Mcm4,6,7 unwinding stops at a nick in either strand. The Mcm4,6,7 ring also actively translocates along duplex DNA, enabling the protein to drive branch migration of Holliday junctions. The Mcm4,6,7 mechanism is very similar to DnaB, except the proteins translocate with opposite polarity along DNA. Mcm4,6,7 and DnaB have different structural folds and evolved independently; thus, the similarity in mechanism is surprising. We propose a "pump in ring" mechanism for both Mcm4,6,7 and DnaB, wherein a single-stranded DNA pump is situated within the central channel of the ring-shaped helicase, and unwinding is the result of steric exclusion. In this example of convergent evolution, the "pump in ring" mechanism was probably selected by eukaryotic and bacterial replication fork helicases in order to restrict unwinding to replication fork structures, stop unwinding when the replication fork encounters a nick, and actively translocate along duplex DNA to accomplish additional activities such as DNA branch migration.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.     

DnaB helicase activity is modulated by DNA geometry and force

The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.     

Escherichia coli DNA helicases: mechanisms of DNA unwinding

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.     

Mechanistic and biological aspects of helicase action on damaged DNA

Helicases catalytically unwind structured nucleic acids in a nucleoside-triphosphate-dependent and directionally specific manner, and are essential for virtually all aspects of nucleic acid metabolism. ATPase-driven helicases which translocate along nucleic acids play a role in damage recognition or unwinding of a DNA tract containing the lesion. Although classical biochemical experiments provided evidence that bulky covalent adducts inhibit DNA unwinding catalyzed by certain DNA helicases in a strand-specific manner (i.e., block to DNA unwinding restricted to adduct residence in the strand the helicase translocates), recent studies suggest more complex arrangements that may depend on the helicase under study, its assembly in a protein complex, and the type of structural DNA perturbation. Moreover, base and sugar phosphate backbone modifications exert effects on DNA helicases that suggest specialized tracking mechanisms. As a component of the replication stress response, the single-stranded DNA binding protein Replication Protein A (RPA) may serve to enable eukaryotic DNA helicases to overcome certain base lesions. Helicases play important roles in DNA damage signaling which also involve their partnership with RPA. In this review, we will discuss our current understanding of mechanistic and biological aspects of helicase action on damaged DNA.     

An ancient anion‐binding structural module in RNA and DNA helicases

RNA and DNA helicases manipulate or translocate along single strands of nucleic acids by grasping them using a conserved structural motif. We have examined the available crystal structures of helicases of the two principal superfamilies, SF1 and SF2, and observed that the most conserved interactions with the nucleic acid occur between the phosphosugar backbone of a trinucleotide and the three strand‐helix loops within a (β‐strand/α‐helix)₃ structural module. At the first and third loops is a conserved hydrogen‐bonded feature called a thr‐motif, often seen at α‐helical N‐termini, with the threonine as the N‐cap residue. These loops can be aligned with few insertions or deletions, and their main chain atoms are structurally congruent amongst the family members and between the two modules found as tandem pairs in all SF1 and SF2 proteins. The other highly conserved interactions with nucleic acid involve mainchain NH groups, often at the helical N‐termini, interacting with phosphate groups. We comment on how the sequence motifs that are commonly used to identify helicases map to locations on the module and discuss the implications of the conserved orientation of nucleic acid on the surface of the module for directional stepping along DNA or RNA. Proteins 2010. © 2010 Wiley‐Liss, Inc.